In the past decade, the nitrosoureas have gained acceptance as potent antitumor agents (Johnston, et al., J. Med. Chem., 14:600 (1971)). The accepted mode of action appears to be through the release of isocyanate in vivo. The two compounds most frequently used clinically are 1-cyclohexyl-3-(-chloroethyl)-3-nitrosourea(CCNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU) which release in vivo an isocyanate derived from the unnitrosated side of the molecule, and an alkylating agent from the other side.
Numerous studies have been directed toward the metabolic products produced in vivo and in vitro in aqueous media, which consist of mainly 2-chloroethanol, vinyl chloride, acetaldehyde, and dichloroethane (Johnston, et al., J. Med. Chem., 18:634 (1975)). It is also known that the N-nitroso-N-alkyl ureido portion of the molecule alkylates DNA (deoxyribonucleic acid) in vivo and in vitro (Frei, et al., Biochem. J., 174:1031 (1978)). In fact, it has been shown that the carcinogenic effectiveness of agents such as N-methyl-N-nitrosourea correlate with the extent of alkylation of the guanine moiety in DNA of target tissues at the C-6 atom.
Alkylation of DNA occurs within an hour after administration of the nitrosourea, and the half-life of the alkylated products is about 24 to 48 hours ((Reed, et al., (Cancer Res., 35:568 (1975)). The study indicated that low doses of nitrosoureas pose only a small threat as mutagens, and hence, are not significantly carcinogenic. From this it may be postulated that the more transglutaminase-specific (see below) the isocyanate resulting from decomposition of the nitrosourea, the lower the required dose, resulting in a reduced risk of carcinogenesis from the antitumor agent.
It has recently been shown that a number of isocyanates are potent inhibitors of the enzyme transglutaminase (Gross, et al., J. Biol. Chem., 250:7693 (1975)), a calcium-dependent enzyme which catalyzes the lysine-glutamine crosslinking of certain proteins present on neoplastic cell surfaces. This enzyme has been implicated in the uncontrolled proliferation of cancer cells (Yancey and Laki, Ann. N.Y. Acad. Sci., 202:344 (1972)). It has been proposed that these crosslinked proteins form an extracellular coating causing the cell to be unrecognized by the cellular immune system, thus preventing normal destruction of foreign neoplastic tissue. The enzyme is fairly specific toward glutamine residues as substrates, and isocyanates resembling these residues have been found to be the most effective inhibitors (Gross, et al., J. Biol. Chem., 250:7693 (1975)).
The structure of the active site of the transglutaminase has been found to contain the pentapeptide sequence --Tyr--Gly--Gln--Cys--Trp-- and has the shape of a pocket approximately 5.times.5 Angstroms (Folk and Cole, J. Biol. Chem., 241:3238 (1966)).